Stereochemical Outcome at Four Stereogenic Centers during

Jul 5, 2012 - Action and Timing of BacC and BacD in the Late Stages of Biosynthesis of the Dipeptide Antibiotic Bacilysin. Jared B. Parker and Christo...
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Stereochemical Outcome at Four Stereogenic Centers during Conversion of Prephenate to Tetrahydrotyrosine by BacABGF in the Bacilysin Pathway Jared B. Parker and Christopher T. Walsh* Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, United States S Supporting Information *

ABSTRACT: The first four enzymes of the bacilysin antibiotic pathway, BacABGF, convert prephenate to a tetrahydrotyrosine (H4Tyr) diastereomer on the way to the anticapsin warhead of the dipeptide antibiotic. BacB takes the BacA product endocyclic-Δ4,Δ8-7R-dihydrohydroxyphenylpyruvate (enH2HPP) and generates a mixture of 3E- and 3Z-olefins of the exocyclicΔ3,Δ5-dihydrohydroxyphenylpyruvate (ex-H2HPP). The NADH-utilizing BacG then catalyzes a conjugate reduction, adding a pro-S hydride equivalent to C4 to yield tetrahydrohydroxyphenylpyruvate (H4HPP), a transamination away (via BacF) from 2S-H4Tyr. Incubations of the pathway enzymes in D2O yield deuterium incorporation at C8 from BacA and then C9 from BacB action. By 1H NMR analysis of samples of H4Tyr, the stereochemistry at C4, C8, and C9 can be assigned. BacG (followed by BacF) converts 3E-ex-H2HPP to 2S,4R,7R-H4Tyr. The 3Z isomer is instead reduced and transaminated to the opposite diastereomer at C4, 2S,4S,7R-H4Tyr. Given that bacilysin has the 2S,4S stereochemistry in its anticapsin moiety, it is likely that the 2S,4S-H4Tyr is the diastereomer “on pathway”. NMR determination of the stereochemistry of the CHD samples at C8 and C9 allows assignment of all stereogenic centers (except C3) in this unusual tetrahydro-aromatic amino acid building block, giving insights into and constraints on the BacA, BacB, and BacG mechanisms.

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he dipeptide antibiotic bacilysin, produced by strains of Bacillus subtilis, consists of L-alanine in amide linkage with the nonproteinogenic amino acid anticapsin.1 Anticapsin is 3epoxycyclohexanonyl-alanine and bears the reactive epoxyketone warhead that covalently captures the active site cysteine thiolate in the glutaminase domain of the enzyme glucosamine-6phosphate synthase.2 Blockade of this enzyme shuts down production of GlcNAc units required for both bacterial and fungal cell wall assembly.3,4 The producing B. subtilis ligates anticapsin to L-Ala to protect itself and then exports the resultant bacilysin as a prodrug. Uptake by a dipeptide permease transporter of a neighboring microbial cell followed by dipeptidase action liberates anticapsin in a neighboring victim cell (Figure 1). The biosynthetic pathway to anticapsin, and then bacilysin, has been studied genetically to reveal a cluster of five bacA−E genes,5,6 and more recently at the enzyme level to decipher the molecular logic for construction of the epoxycyclohexanone moiety of this unusual amino acid.7 The biochemical studies have shown that ywf GH, the latter transcribed on the opposite DNA strand from bacA−E, encode an NADH-dependent reductase (ywf H) and an L-specific transaminase (ywf G) that carry out steps 3 and 4 of the pathway. Therefore, we rename the genes bacF (ywf G) and bacG (ywf H) and use that nomenclature throughout this work (Figure 2). In sum, the set of four enzymes BacABGF, in that order, act in tandem to divert some of the cellular flux of prephenate away from Phe and Tyr biosynthesis © 2012 American Chemical Society

and into the anticapsin pathway, yielding tetrahydrotyrosine (H4Tyr) as an intermediate.7 We have previously determined that BacA decarboxylates prephenate without aromatization7 and in the process specifically isomerizes the pro-R double bond in the 1,4-diene system to yield endocyclic-Δ4,Δ8-7R-dihydrohydroxyphenylpyruvate (enH2HPP).8 The next enzyme, BacB, conducts an allylic isomerization to move the double bond into conjugation at the exocyclic Δ3-position (ex-H2HPP). In so doing, it generates a 3/ 1 mixture of the E- and Z-geometric isomers at the Δ3-olefin upon equilibration. The third enzyme, BacG, uses NADH to reduce that conjugated exocyclic double bond and yield an H4HPP product.7 The fourth enzyme, BacF, is a transaminase and uses L-Phe as an amino donor to reductively aminate the 2keto group of H4HPP and yields an H4Tyr diastereomer (Figure 2). The H4Tyr is presumably two steps away, epoxidation of the olefin and oxidation of the C7-OH group to the ketone, from anticapsin. H4Tyr, an unusual noncanonical amino acid, has three stereocenters: C2, C4, and C7. As shown below, BacF action, with L-Phe as the cosubstrate, sets the S-stereochemistry at C2, and we have previously established8 that BacA sets R-stereochemistry at C7, which is carried through to H4Tyr. Received: May 16, 2012 Revised: June 21, 2012 Published: July 5, 2012 5622

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Figure 1. Ligation of L-alanine and anticapsin yields the dipeptide antibiotic bacilysin. The host cell further self-protects from anticapsin and bacilysin toxicity by transporting it into the extracellular environment, where it can be imported by neighboring cells. Peptide bond cleavage of bacilysin releases the anticapsin warhead, which blocks cell wall biosynthesis via inhibition of glucosamine-6-phosphate synthase.

Figure 2. Proposed pathway for the biosynthesis of anticapsin from prephenate. YwfG and YwfH are hereby renamed BacF and BacG, respectively, as noted in the inset.

H and two-dimensional NMR spectra were recorded at 25 °C on a Varian VNMRS 600 MHz spectrometer equipped with a triple-resonance probe. 13C one-dimensional NMR spectra were recorded on a Varian MR 400 MHz spectrometer (100.497 MHz for 13C) equipped with a OneNMR probe. NMR data were analyzed and integrated with ACD/Laboratories software. Highresolution LC−MS data were collected on an Agilent Technologies 6520 Accurate-Mass Q-TOF LC−MS system and analyzed using its accompanying software. HPLC was performed on a Beckman Coulter System Gold instrument, and peaks were analyzed using the accompanying Karat 32 software. UV−vis measurements were collected using a Cary 50 BIO UV− vis spectrometer and analyzed with the accompanying software. Cloning, Expression, and Purification of the Enzymes BacABGF. The cloning of plasmids for production of BacABGF has been previously described.7 The expression and purification of BacA and BacB were also performed as previously described.8 The expression and purification of BacG and BacF were identical to the procedure for BacA. Protein concentrations of BacABGF were determined by UV−vis absorbance using the following extinction coefficients (ε280) calculated from the protein primary sequence using the ExPASy Bioinformatics Research Portal: 17420 M−1 cm−1 for BacA, 24410 M−1 cm−1 for BacB, 9970 M−1 cm−1 for BacG, and 41830 M−1 cm−1 for BacF. Generation and Purification of Protonated 3Z-7R- and 3E-7R-ex-H2HPP Isomers for Use as Substrates for NMR and Kinetic Analyses of BacG Action. Conversion of barium prephenate to potassium prephenate was performed as previously described.7 Protonated 3E-ex-H2HPP was generated 1

At issue then is the stereochemistry at C4, presumably established by the NADH-utilizing BacG. Also of note is the fact that while the protio version of H4Tyr has the three previously mentioned stereogenic centers, if the BacA, BacB, and BacG incubations starting from prephenate are conducted in D2O buffers, then one deuterium (D = 2H) is incorporated at C8 via the action of BacA, one at C9 from the action of BacB, and then another incorporated at C3 by BacG catalysis (the hydride from NADH is added at C4).7 Thus, the C3, C8, and C9 methylene groups (-CH2-) become chiral by virtue of deuterium substitution (-CHD-). Therefore, in 2H3-, 2H8-, and 2H9containing samples, H4Tyr produced via the BacABGF pathway possesses six chiral centers: C2−C4 and C7−C9. In this study, we report delineation of stereochemistry at the four centers (C2, C4, C8, and C9) that provide mechanistic insights and constraints into the action of BacF, BacG, BacA, and BacB, respectively.

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MATERIALS AND METHODS Materials and Instrumentation. Prephenic acid barium salt, L- and D-tyrosine (Tyr), L- and D-phenylalanine (Phe), 4hydroxyphenylpyruvic acid (HPP), β-nicotinamide adenine dinucleotide hydrate (NAD+), and β-nicotinamide adenine dinucleotide reduced disodium salt hydrate (NADH) were purchased from Sigma-Aldrich. NMR solvent (D2O), D-glucose (1-D, 98%), and ethanol-d6 (D, 99%) were purchased from Cambridge Isotope Laboratories. The enzymes glucose dehydrogenase from Pseudomonas sp., alcohol dehydrogenase from Saccharomyces cerevisiae, and aldehyde dehydrogenase from S. cerevisiae were also purchased from Sigma-Aldrich. 5623

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phosphate buffer (pH 8.0) in a volume of 250 μL. NADH (2 mM) was found to be >90% saturating for the reactions of both isomers. Reactions were initiated via addition of BacG, and the progress of the reaction was monitored by observing the oxidation of NADH to NAD+ (UV absorbance at 370 nm) in a 1 mm cuvette. Reaction velocities (kobs) in units of absorbance per minute were determined by calculating the slope of the initial, linear region of the 370 nm time course of each reaction. The change in NADH absorbance at 370 nm was converted to concentration using the experimentally determined extinction coefficient ε370 of 2690 M−1 cm−1 (referenced to NADH concentration determined from the ε340 of 6220 M−1 cm−1 10). Data were fit to the Michaelis−Menten equation (eq 1) using GraphPad Prism

in reaction mixtures containing 12 mM potassium prephenate, 4 μM BacA, and 1 μM BacB in 2 mL of potassium phosphate buffer (pH 8.0). The reaction mixtures were incubated at ∼20 °C for 16 h. The reactions were quenched via addition of acetonitrile to a concentration of 30% (v/v), and the mixtures were frozen and lyophilized in preparation for purification (discussed after generation of 3Z-ex-H2HPP). Production and purification of large amounts of 3Z-ex-H2HPP from BacAB reactions were found to be very laborious because of a maximum of only 25% of the total product being the 3Z isomer.8 However, NMR and kinetic experiments with the enzymes AerD and AerE (BacA and BacB homologues, respectively, from Planktothrix agardhii9) showed that these enzymes produce products identical to those of BacAB action when acting on prephenate substrate, except the 3Z/3E isomer ratio is 9/1 (J. B. Parker and C. T. Walsh, unpublished data) as opposed to 1/3 for BacAB. Thus, fully protonated 3Z-ex-H2HPP was generated from AerDE reaction mixtures containing 12 mM potassium prephenate, 2 μM AerD, and 4 μM AerE in 4 mL of 50 mM potassium phosphate buffer (pH 8.0). {It is important to note that formation of the 3Z isomer from either AerE or BacB action was severely inhibited by high concentrations of salt [approximately >250 mM (data not shown)].} The reaction mixtures were incubated at ∼20 °C for 16 h; the reactions were quenched via addition of acetonitrile to a final concentration of 30% (v/v), and the mixtures were frozen and lyophilized to dryness. For purification of the ex-H2HPP isomers, the lyophilized reaction mixtures of BacAB (for 3E isomer formation) and AerDE (for 3Z isomer formation) were resuspended (separately) in 750 μL of 10 mM potassium phosphate buffer (pH 8.0) and loaded onto a 100 mm × 21.2 mm Hypercarb column (Thermo Scientific) equilibrated in the resuspension buffer. Bound compounds were eluted (3Z and 3E isomers separated) by exposure to a shallow linear gradient of acetonitrile. Compound elution was monitored by UV absorbance at 295 nm. Fractions containing the appropriate isomers (normally ∼30 mL) were pooled, frozen, and lyophilized to dryness. Pure ex-H2HPP isomers were then moderately desalted by being resuspended in 500 μL of 10 mM potassium phosphate buffer (pH 8.0) and loaded onto a 100 mm × 10 mm Hypercarb column equilibrated in the same buffer. Bound ex-H2HPP was eluted with a sharp linear gradient of acetonitrile (0 to 90% over 25 min), and 4.5 mL of fractions containing ex-H2HPP were pooled and lyophilized to dryness. The lyophilized compound and residual potassium phosphate buffer were resuspended in 300 μL of ddH2O, and the concentration was determined by UV−vis absorbance [ε295 = 15300 M−1 cm−1 for the 3E isomer,7 and ε295 = 13700 M−1 cm−1 for the 3Z isomer (Figure S10 of the Supporting Information)]. Isomer purity was analyzed via analytical HPLC using a 100 mm × 2.1 mm Hypercarb column equilibrated in 10 mM potassium phosphate buffer (pH 8.0). Bound isomers were eluted using a linear gradient of acetonitrile, and elution was monitored by UV−vis absorbance at 295 nm (Figure S1 of the Supporting Information). Isomers were stored at −80 °C to prevent nonenzymatic isomer equilibration when not in use. Kinetics of the Action of BacG on 3Z- and 3E-ex-H2HPP. Kinetic reaction mixtures for the analysis of the action of BacG on 3Z-ex-H2HPP contained 1 μM BacG, 2 mM NADH, 0−1.1 mM 3Z-ex-H2HPP, and 50 mM potassium phosphate buffer (pH 8.0) in a volume of 250 μL. Kinetic reaction mixtures for the analysis of the action of BacG on 3E-ex-H2HPP contained 5 μM BacG, 2 mM NADH, 0−8 mM 3E-ex-H2HPP, and 50 mM potassium

kobs =

kcat[S] K m + [S]

(1)

where kcat is the maximal velocity, [S] is the substrate concentration, and Km is the substrate concentration that yields kobs = 1/2kcat. All reactions were performed in triplicate except the reaction that contained 8 mM 3E-ex-H2HPP, which was performed in duplicate because of a lack of material. Generation and Purification of Protonated 4S- and 4RH4HPP and 4S- and 4R-H4Tyr Isomers for NMR Analysis. 4S-H4HPP was generated in a reaction mixture containing 2 mM 3Z-ex-H2HPP, 5 mM NADH, and 20 μM BacG incubated in 1 mL of 50 mM potassium phosphate buffer (pH 8.0). 4S-H4Tyr was generated in a reaction mixture containing ∼2 mM 4SH4HPP (described in the previous sentence), 10 mM L-Phe, 0.1 mM PLP, and 20 μM BacF in 1 mL of 50 mM potassium phosphate buffer (pH 8.0). 4R-H4HPP was generated in a reaction mixture containing 2 mM 3E-ex-H2HPP, 5 mM NADH, and 20 μM BacG in 1 mL of 50 mM potassium phosphate buffer (pH 8.0). 4R-H4Tyr was generated in a reaction mixture containing ∼2 mM 4R-H4HPP (described in the previous sentence), 10 mM L-Phe, 0.1 mM PLP, and 20 μM BacF in 1 mL of 50 mM potassium phosphate buffer (pH 8.0). All reaction mixtures were incubated at ∼20 °C for 16 h. All of the reactions described above were quenched via addition of acetonitrile to a final concentration of 30% (v/v), and the mixtures were frozen and lyophilized to dryness. Dried reaction mixtures were resuspended in 500 μL of 10 mM potassium phosphate buffer (pH 8.0) and purified as described above for the ex-H2HPP isomers except using a 100 mm × 10 mm Hypercarb column. As H4HPP and H4Tyr do not have a characteristic UV−vis absorbance, their elution was monitored via LC−MS in negative detection mode (0.1% NH4OH as the solvent additive) using a 50 mm × 2.1 mm Hypercarb column with ddH2O as the mobile phase and acetonitrile as the eluent. A mass of 183.0668 was observed for the H4HPP isomers (calcd, 183.0663) and 184.0986 (calcd, 184.0979) for the H4Tyr isomers. Three milliliters of fractions containing the desired product were frozen and lyophilized to dryness, resuspended in 300 μL of 99.99% D2O, and placed in a 5 mm D2O matched Shigemi tube for NMR analysis. NMR spectral assignments were made via analyses of 1H, 13C, 1 H−1H gCOSY, 1H−13C gHSQC, 1H−13C gHMBC, and 1 H−1H NOESY (500 ms mixing time) spectra. Proton spectra were referenced to residual H2O (4.79 ppm), while 13C spectra were referenced to the methyl carbon of acetonitrile (1.47 ppm).11 [Acetonitrile was spiked into the NMR sample at 0.3% 5624

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All deuterated H4HPP and H4Tyr production reaction mixtures were incubated at ∼20 °C for 16 h and then reactions quenched and mixtures purified; appropriate fractions were identified by LC−MS as for the protonated H4HPP and H4Tyr reactions described above. H4HPP masses obtained were 185.0816, 186.0876, 187.0938, and 188.0993 (calcd, 185.0788, 186.0851, 187.0914, and 188.0977). H4Tyr masses obtained were 186.1159, 187.1215, 188.1279, and 189.1339 (calcd, 186.1105, 187.1167, 188.1230, 189.1293). These masses correspond to compounds with incorporation of two, three, four, and five deuteriums, respectively. Enzymatic Synthesis of 4R-2H- and 4S-2H-NADH. 4R-2HNADH (also known as A-side NADD) was synthesized following a modified protocol of Viola et al.12 Ethanol-d6 (25 mM), 10 mM NAD+, 300 units of alcohol dehydrogenase, and 32 units of aldehyde dehydrogenase were mixed in 10 mL of 25 mM Taps buffer (pH 9.0). The pH of the buffer was readjusted to 9.0 after addition of the NAD+ but before addition of enzymes. The reaction mixture was incubated at ∼20 °C, and reaction progress was monitored by following the appearance of NADH (UV absorbance at 340 nm). After the mixture had been incubated for 45 min, the reaction was quenched via protein removal using a 3500 MWCO centrifugal filtration device (Amicon). 4S-2H-NADH (also known as B-side NADD) was synthesized according to a slightly modified protocol of Ottolina et al.13 Glucose-d1 (30 mM), 15 mM NAD+, and 100 units of glucose dehydrogenase were mixed in 6 mL of 50 mM potassium phosphate buffer (pH 7.5). The pH of the buffer was readjusted to 7.5 before enzyme addition. Reaction progress was monitored, and the reaction was quenched (after an 80 min reaction time) exactly as for the A-side NADD synthesis reaction described above. The processing of both NADD reactions was identical for the remaining steps. The postfiltration flow-through containing the NADD was frozen and lyophilized to dryness. The dried reaction components were resuspended in 1 mL of H2O. NADD at this stage was used directly in the BacG enzymatic reactions after the pH had been corrected to 8.0. The NADD concentration was determined by UV absorbance (ε340 = 6220 M−1 cm−1 10). To prepare NADD samples for NMR analysis of the deuterium content at C4, NADD was crudely purified by being loaded onto a 250 mm × 10 mm Luna C18 5 μm HPLC column (Phenomenex) equilibrated in H2O and 0.01% NH4OH. Bound NADD was eluted using a linear gradient of acetonitrile spiked with 0.01% NH4OH. Fractions containing NADD were identified by the characteristic absorbance maxima at 260 and 340 nm. The observed 260 nm/340 nm ratio was 2.3 for A-side and 2.5 for B-side NADD (previously reported as ∼2.2 − 2.3 for pure NADH12). Fractions containing NADD were pooled and lyophilized to dryness. Samples (2 mM) of both A-side and Bside NADD were prepared in 99.99% D2O and placed in 5 mm D2O matched Shigemi tubes for 1H NMR analysis (Figure S2 of the Supporting Information). Pro-R and pro-S hydride peaks were identified on the basis of previous assignments.13,14 Enzymatic Synthesis of H4HPP Isomers from BacG Action with 4R-2H-NADH and 4S-2H-NADH as Cosubstrates. Reaction mixtures for the generation of 4S- and 4RH4HPP from protonated 3Z- and 3E-ex-H2HPP, respectively, contained 1 mM ex-H2HPP, 2 mM 2H-NADH, and 30 μM BacG in 1 mL of H2O containing 50 mM potassium phosphate buffer (pH 8.0). The reaction mixtures were incubated, the reactions quenched, the mixtures purified, and the fractions identified exactly as described above for protonated H4HPP isomers

(v/v) after all necessary spectra had been acquired, and an additional 13C experiment was performed to obtain the reference so as not to contaminate the original spectra with the acetonitrile signal.] Unless otherwise stated, water suppression (via presaturation) was utilized in all proton spectra that were collected. Generation and Purification of Deuterated 3Z-exH2HPP and 3E-ex-H2HPP from the Action of BacA and BacB and Deuterated 3E-ex-H2HPP from the Action of Only BacA. The reaction mixtures for the production of deuterated ex-H2HPP isomers from the action of BacAB contained 5 mM prephenate, 10 μM BacA, and 40 μM BacB in 1 mL of 95% D2O containing 50 mM potassium phosphate (pD 8.0). The reaction mixtures were incubated at 37 °C for 24 h; the reactions were quenched, and the mixtures were purified and desalted as described above for the protonated ex-H2HPP isomers. (BacAB reactions in D2O were very inefficient at producing 3Z-ex-H2HPP. Therefore, it took many of these reactions to obtain enough deuterated compound for NMR analysis.) After desalting and lyophilization, the purified deuterated 3Z- and 3E-ex-H2HPP isomers were resuspended in D2O, and 1H, 1H−13C gHSQC, and 1H−1H NOESY data were collected and referenced as described above for the protonated H4HPP isomers. Production of deuterated 3E-ex-H2HPP from BacA action (without BacB) in tandem with nonenzymatic diene isomerization was accomplished in a reaction mixture containing 10 mM prephenate and 10 μM BacA in 2 mL of 98% D2O containing 50 mM potassium phosphate buffer (pD 8.0). The reaction mixture was incubated at 37 °C for 144 h. The progress of the extremely lethargic nonenzymatic isomerization in D2O was monitored by UV absorbance (λmax of en-H2HPP = 258 nm; λmax of ex-H2HPP = 295 nm7). The reaction was then quenched; the mixture was purified and desalted, and 1H and 1H−13C gHSQC NMR data were collected as described above for the BacAB-generated deuterated ex-H2HPP isomers. Enzymatic Synthesis of Deuterated H4HPP Isomers and H4Tyr Isomers in D2O Solvent. Two forms of deuterated 4R-H4HPP were produced in two distinct reactions. The first reaction mixture contained 1 mM deuterated 3E-ex-H2HPP produced from the deuterated BacAB reaction (described above), 4 mM NADH, and 30 μM BacG in 1 mL of 97% D2O containing 50 mM potassium phosphate buffer (pD 8.0). The second reaction mixture contained 5 mM potassium prephenate and 20 mM NADH and was simultaneously incubated with 20 μM BacABG in 95% D2O containing 50 mM potassium phosphate buffer (pD 8.0). Likewise, two forms of deuterated 4R-H4Tyr were produced in two distinct reactions. The first reaction mixture contained 2 mM deuterated 3E-ex-H2HPP produced from BacAB action (described above), 8 mM NADH, 0.1 mM PLP, 10 mM L-Phe, and 15 μM BacGF in 1 mL of 97% D2O containing 50 mM potassium phosphate buffer (pD 8.0). The second reaction mixture contained 5 mM potassium prephenate, 20 mM NADH, 20 mM L-Phe, 0.1 mM PLP, and 15 μM BacABGF in 95% D2O containing 50 mM potassium phosphate buffer (pD 8.0). Only one form of deuterated 4S-H4HPP and 4S-H4Tyr could be produced. The reactions for the production of deuterated 4SH4HPP and 4S-H4Tyr were identical to the first set of reactions used to produce deuterated 4R-H 4 HPP and 4R-H 4 Tyr (described in the previous paragraph) except that deuterated 3Z-ex-H2HPP (produced from the deuterated BacAB reaction described above) was used in place of 3E-ex-H2HPP. 5625

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Figure 3. Michaelis−Menten kinetic traces for the reductase action of BacG on (A) 3Z-ex-H2HPP (kcat = 71.3 min−1; Km = 0.09 mM) and (B) 3E-exH2HPP (kcat = 33.2 min−1; Km = 2.7 mM) in the presence of a saturating level of NADH (2 mM).

Figure 4. 1H NMR traces of purified (A) 4S-H4HPP from the action of BacG on 3Z-ex-H2HPP, (B) 4S-H4Tyr from the action of BacF on panel A, (C) 4R-H4HPP from the action of BacG on 3E-ex-H2HPP, and (D) 4R-H4Tyr from the action of BacF on panel C.

Preparation of Tyrosine from the Transaminase Action of BacF on 4-Hydroxyphenylpyruvate for the Determination of Amino Acid Chirality. HPP (2 mM), 10 mM Phe (both L and D enantiomers in separate reactions), 0.5 mM PLP, and 10 μM BacF were mixed in 1 mL of 50 mM potassium phosphate buffer (pH 8.0). Reaction mixtures were incubated at ∼20 °C for 16 h, reactions quenched via addition of acetonitrile to a final concentration of 30% (v/v), and mixtures lyophilized to

generated from protonated NADH. The detected LC−MS mass of H4HPP produced from 4R-2H-NADH was 183.0670 (calcd, 183.0663). A major mass (184.0726) and a minor mass (183.0666) were both detected from H4HPP produced from 4S-2H-NADH (calcd, 184.0726 and 183.0663, respectively). Collection of 1H and 1H−13C gHSQC NMR data was also performed as described above (Figure S3 of the Supporting Information). 5626

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dryness and resuspended in 500 μL of H2O. The expected transamination product (tyrosine) was identified from the BacF reaction mixture containing L-Phe by LC−MS in positive detection mode (0.1% formic acid as a solvent additive) using a 50 mm × 2.1 mm Hypercarb column with water as the mobile phase and acetonitrile as the eluent. No tyrosine was observed from the BacF reaction mixture containing D-Phe as the cosubstrate. Prior to chiral HPLC analysis of the BacF-produced tyrosine, it was necessary to purify the nascent tyrosine from the other reaction components to avoid spectral contamination. The resuspended reaction mixture was loaded onto a 100 mm × 10 mm Hypercarb column equilibrated in 10 mM potassium phosphate buffer (pH 8.0). Bound compounds were eluted via a linearly increasing gradient of acetonitrile. Fractions containing tyrosine were initially located by their UV−vis spectra and later confirmed by LC−MS analysis as described above. The appropriate fractions were pooled and lyophilized to dryness. The amino acid chirality of the BacF-generated tyrosine was determined by comparing its elution profile from a 150 mm × 4.6 mm Chirex 3126 column (Phenomenex) equilibrated in 2 mM copper(II) sulfate in an 84/16 (v/v) water/methanol mixture with that of authentic L- and D-tyrosine standards. Tyrosine elution was monitored by UV−vis absorbance at 280 nm (Figure S4 of the Supporting Information).

efficiencies of two substrates for the same enzyme, the 3Z,7R-exH 2 HPP isomer is a 64-fold better substrate than the corresponding E isomer. Thus, although the 3Z geometric isomer is kinetically disfavored during BacB action, and essentially absent from the nonenzymatic isomerization of enH2HPP to the thermodynamically favored ex-H2HPP,8 it is very much the preferred substrate for the conjugate reduction reaction of BacG. Panels A and C of Figure 4 show the proton NMR spectra of the H4HPP samples generated from the action of BacG on 3Z-exH2HPP and 3E-ex-H2HPP substrates, respectively. It is clear from examination of the H8 and H9 resonances that the products are distinct, most likely because of distinct diastereomers at C4, the anticipated site of hydride addition by BacG. On subsequent BacF-mediated transamination of each of the H4HPP keto acid samples to the corresponding 2S-H4Tyr samples, the proton NMR spectra in panels B (derived from the Figure 4A sample) and D (derived from the Figure 4C sample) of Figure 4 also gave distinct diastereomers as evidenced by the distinct splitting patterns from the methylene hydrogens at C3, C8, and C9. The assignments of the indicated stereochemistries as 4S (from the 3Z substrate) and 4R (from the 3E substrate) are detailed in a subsequent section. Proof that BacG acted by conjugate addition of an NADHderived hydride to C4 of the ex-H2HPP substrates was explicitly tested with 4R-D1-NADH and 4S-D1-NADH samples as reductants. 1H NMR analysis of the two chirally deuterated NADH samples established that 4R-D1-NADH was 40% D1 while 4S-D1-NADH was 62% D1 (Figure S2 of the Supporting Information). NMR analysis of the H4HPP products from 4R-D1-NADH incubations on both the 3E- and 3Z-ex-H2HPP substrates shows the transfer of the diastereotopic 4-pro-S hydride to both the 3E and 3Z substrate isomers. Figure S3 of the Supporting Information shows the 3Z substrate had 0.94 equiv of 1H at C4 while the 3E substrate possessed 1.0 equiv of 1H, referenced to the single hydrogen at C7 in the product H4HPPs. One could worry that the lack of deuterium reflected a kinetic isotope effect between NADH and 4R-D1-NADH molecules in the NADH reductant pool, but the complementary results with the transfer of deuteride from the 4S-D1-NADH sample ruled out that possible ambiguity. The 4S-D1-NADH donor sample (62% deuterium content) gave H4HPP with 68% D at C4 when the 3Z substrate was reduced and 58% D at C4 when the 3E substrate was reduced. These results clearly indicate that BacG transfers the pro-S hydride of NADH during ex-H2HPP reduction. These results validate several points. First, BacG is acting as a regiospecific hydride transfer catalyst to C4 for each of the 3Eand 3Z-ex-H2HPP substrate isomers. Second, the pro-S hydride of NADH is transferred to each of the substrate geometric isomers. Third, from the NMR spectra in panels A and C of Figure 4 noted above, the H4HPP products are likely to be diastereomers at C4. The BacF Transaminase Is 2S-Specific. Given that the BacF transaminase processes L-Phe (2S) as an amino donor to cosubstrate H4HPP, we anticipated it would show the same specificity toward partner keto acids that become reductively aminated. To that end, we have now demonstrated that while LPhe is an effective amino donor, D-Phe is not (data not shown). To validate that the keto acid partner undergoing amination gives a 2S amino acid product, we used p-hydroxyphenylpyruvate as the acceptor with L-Phe as a donor. As anticipated, tyrosine was generated (LC−MS mass of 182.0805; calcd, 182.0812) as L-Phe

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RESULTS BacG Acts on 3Z,7R- and 3E,7R-ex-H2HPP Geometric Isomers To Give H4HPP Products That Are Diastereomeric at C4. In initial studies of the Bac pathway enzymes for the conversion of prephenate to H4Tyr, we conducted incubations of either BacAGF or BacABGF in parallel, based on the observation that the BacA-derived en-H2HPP will isomerize nonenzymatically and quantitatively into conjugation to give the exocyclic exH2HPP product.7 BacB accelerates the allylic isomerization by up to 103 and so was of use in shortening the kinetics in the four enzyme incubations.15 These incubations could be conducted in buffered H2O or buffered D2O, to yield H4Tyr with one deuterium each at C8 and C9 [as well as at C2 and C3 (vide infra)]. A deeper analysis of the BacB-mediated isomerization recently led us to the finding that while the E/Z ratio of ex-H2HPP from BacA followed by nonenzymatic allylic isomerization was 50/1, in BacB-containing equilibrated incubations the E/Z ratio was 3/ 1. In turn, that led us to separation and structural assignment of the 3E and 3Z isomers on Hypercarb HPLC columns and a proposed mechanism for geometric isomer equilibration via dienolate intermediates.8 The presumption from the above results was that each of the 3E- and 3Z,7R-ex-H2HPP geometric isomers could be a substrate for the NADH-utilizing BacG. This enzyme is assayed by loss of the 340 nm chromophore of the reduced nicotinamide ring of NADH as ex-H2HPP is reduced to H4HPP (and NADH is oxidized to NAD+). Because of the partially overlapping chromophores of the ex-H2HPP substrate (λmax = 295 nm) and cosubstrate NADH, measurements of the reaction were taken away from the absorbance maximum of NADH, at 370 nm, where a sufficient signal-to-noise ratio persisted. Indeed, the separated geometric 3E (96% purity) and 3Z [97% purity (Figure S1 of the Supporting Information)] isomers were each saturable substrates (Figure 3). The kcat for the 3Z isomer at 71.3 min−1 was approximately twice that for the 3E isomer (33.2 min−1). The 3Z isomer also had a 30-fold lower Km value (0.09 mM vs 2.7 mM). By kcat/Km ratios, which compare the relative catalytic 5627

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Figure 5. 1H−1H NOESY spectra and accompanying NOE correlations used to determine the C4 stereochemistry of (A) 4S-H4Tyr and (B) 4R-H4Tyr. Because of the spectral overlap of H3a, H8a, and H9b in the spectra of (B) 4R-H4Tyr, NOE correlations of (C) 4R-H4Tyr-d4 were also analyzed to allow analysis of correlations to and from H9b to complete the stereochemical determination. Black cross-peaks in the NOESY spectra indicate positive resonances, while red cross-peaks indicate negative resonances. Key NOE interactions have been noted on the structures with dashed lines, and the correlating cross-peaks are boxed in the NOESY spectra.

was oxidatively deaminated in the first half of the BacF reaction. The chirality of the nascent Tyr was determined to be “L” (2S) by comparison with authentic D- and L-Tyr samples by chiral HPLC

(Figure S4 of the Supporting Information). By analogy, we assign the H4Tyr from reductive amination of H4HPP by BacF as the 2S diastereomer. At this point, with the 2S stereochemistry assigned, 5628

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Figure 5B by the strong correlation between H8b and H9a). Additional support for these analyses comes from the 1H−1H NOESY spectra of protonated 4R-H4HPP spectra (Figure S9 of the Supporting Information), where H8a is a distinct resonance. In this spectrum, strong correlations are seen between H8a and the protons on C4 and C7 (same face) while little or no correlations are seen between H9a and either H4 or H7 (opposite face). Stereochemical Insights at C8 and C9 in Deuterated Samples of H4HPP and H4Tyr. A bonus from NMR analyses of the deuterated 4R- and 4S-H4Tyr samples (as seen in Figure 5C and Figures S5B, S6C, and S7C of the Supporting Information) is information about the placement of deuterium at C8 (from BacA action) and C9 (from BacB action). We know the stereochemistry at C2 is 2S from BacF transaminase action data cited above. We have not tried to determine the stereochemistry at the C3-HD center. One deuterium is picked up at that site during BacG action, but there is also solvent exchange at C3 when BacB conducts the equilibration between the 3E and 3Z isomers of ex-H2HPP, via dienolate intermediates.8 The extent of that exchange may well have differed during different incubations, leading to partial deuterium incorporations at C3. At C8, the deuterium in both the 4R- and 4S-H4Tyr products is on the same face as H7; therefore, the stereochemistry in the C8HD samples is 8S. The stereochemical outcome at C9 in the C9HD samples is more complex. As shown in Figure 5 and Figure S5B of the Supporting Information in the 4R-H4Tyr sample from simultaneous BacABGF D2O incubations with prephenate, the deuterium at C9 is on the ring face opposite both H4 and H7 and therefore is the 9S monodeutero isomer. The results with 4S-H4Tyr samples from D2O incubations were less clear-cut. Under some conditions (Figure S7C of the Supporting Information), with BacB present, there was partial deuterium substitution at both the 9S and 9R positions. In experiments where the BacA product en-H2HPP was allowed to isomerize nonenzymatically (in solutions estimated at 98% D2O and then carried through to the BacGF reactions in D2O), there was very little deuterium at C9 (Figure S8A of the Supporting Information), perhaps reflecting a large deuterium isotope effect in the nonenzymic isomerization of en-H2HPP solely to the 3Eex-H2HPP isomer. Because of these vagaries due to BacBmediated exchange and the nonenzymatic isomerization giving solely the 3E-ex-H2HPP product,8 we have not conducted a full interpretation of the C9 results in the 4S-H4Tyr diastereomer.

we turned to analysis of the absolute stereochemistry at C4 of the H4Tyr samples generated from tandem action of BacABGF to obtain the absolute stereochemistry of the H4Tyr emerging from the four enzymes in the bacilysin pathway. Interpretation of NOE Correlations in Determining the Stereochemistry of 4S-H4Tyr and 4R-H4Tyr at C4. To determine the stereochemistry at C4 of H4Tyr (and by inference H4HPP) derived from BacG reduction of 3Z-ex-H2HPP, we collected 1H−1H NOESY data reporting on the relative distances between protons on a fully protonated sample of H4Tyr (Figure 5A). Because the proton resonances needed to make the appropriate correlations did not overlap in this sample, stereochemical assignment was straightforward. We began linking correlations using H7 as the reference, because the stereochemistry at C7 had been previously assigned in our earlier study as R stereochemistry.8 From inspection of the NOE crosspeaks, it is seen that H7 shows a strong correlation with H8a and H9a, but not H8b and H9b. This indicates that H7, H8a, and H9a are on the same face of the cyclohexenol ring, while H8b and H9b are on the opposite face (Figure 5A). We then moved our attention to the H4 proton and noticed that H4 correlates strongly with H8b and H9b, but not H8a and H9a, indicating that H4, H8b, and H9b are on the same face of the cyclohexenol ring and thus on the opposite face from H7. These correlations establish that H4Tyr (and thus H4HPP, as shown in Figure 4) originating from BacG reduction of 3Z-ex-H2HPP possesses 4S stereochemistry (Figure 5A). Determination of the C4 stereochemistry of H4Tyr (and H4HPP) derived from BacG reduction of 3E-ex-H2HPP was not so straightforward because of the overlapping cross-peaks of H3a, H8a, and H9b in the 1H−1H NOESY data of protonated H4Tyr (Figure 5B). However, two important NOE correlations could be clearly seen in these data: a strong cross-peak between H8b and H9a and a weak cross-peak between H4 and H7. While the strong cross-peak between H8b and H9a indicates they are most likely on the same face of the cyclohexenol ring, the NOE correlation between H4 and H7 confirms that H4 and H7 are on the same face of the ring. This shows that 4R-H4Tyr (and thus 4R-H4HPP) is derived from the action of BacG on 3E-ex-H2HPP (Figure 5B). Although the stereochemistry at C4 of 4R-H4Tyr could be interpreted despite the overlapped resonances in the 1H−1H NOESY spectrum (Figure 5B), NOE analysis of 4R-H4Tyr-d4 (Figure 5C) was necessary to determine the relative positions of the nonequivalent protons of C8 and C9. As observed in Figure 5C and in greater detail in Figure S5B of the Supporting Information, simultaneous incubation of BacABGF with prephenate in 95% D2O solvent incorporates one deuterium each at C2, C3, C8, and C9 of 4R-H4Tyr-d4 and fortuitously yields a 1H NMR spectra without overlapped resonances. Inspection of the 1H−1H NOESY data of 4R-H4Tyr-d4 shows an especially strong cross-peak between H4 and H9b, indicating they are on the same face of the cyclohexenol ring. (For comparison, the crosspeak between H4 and H9a in Figure 5B is weaker. The cross-peak between H4 and H7 in panels B and C of Figure 5 can be used to normalize the spectra to each other.) Also in Figure 5C, almost equivalent cross-peak intensities are seen between H7 and H8b and between H7 and H9b. For this to be possible, H8b would need to be on the opposite face of H7 while H9b would need to be on the same face as H7. This correlation agrees with the determination made above that H4 and H9b are on the same face of the cyclohexenol ring (Figure 5C). Combined, these determinations imply that H4, H7, H8a, and H9b are on one ring face while H8b and H9a are on the opposing face (as suggested in

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DISCUSSION Prephenate is the immediate precursor to both phenylpyruvate (via prephenate dehydratase16,17) and hydroxyphenylpyruvate (via prephenate dehydrogenase18,19) on the way to L-Phe, L-Tyr, and many phenylpropanoid metabolites.20,21 In the anticapsin/ bacilysin pathway, prephenate is instead shunted down a newly appreciated metabolic branch. BacA, the first enzyme in this and related pathways to dihydro- and tetrahydro-aromatic metabolite scaffolds,9 is a remarkable catalyst, engineering the decarboxylation without aromatization of the 1,4-cyclohexadiene ring of prephenate.7 It is a regiospecific isomerization catalyst, working only on the pro-R, not the pro-S, double bond of the prephenate diene and delivering a proton only to C6, not C6′, a carbon that becomes C8 in the en-H2HPP product.8 Three enzymes later, by tandem action of BacBGF, H4Tyr is formed. Unlike its familiar aromatic congener L-tyrosine that has one chiral center (2S), H4Tyr has three chiral centers: C2, the ring junction C4, and the original alcoholic carbon of prephenate, C7. 5629

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Figure 6. BacG transfers the pro-S hydride of NADH to opposite faces of (A) 3E-ex-H2HPP and (B) 3Z-ex-H2HPP to produce 4R-H4HPP and 4SH4HPP, respectively. (C) BacA decarboxylation of prephenate performed in D2O solvent produces en-H2HPP with “S” stereochemistry at C6. This indicates that the incoming proton is delivered to the face of the cyclohexadiene ring opposite the departing CO2. (D) Action of BacABGF on prephenate produces 2S,4S,7R-H4Tyr and 2S,4R,7R-H4Tyr, with the relative amounts produced being determined by the action of BacB.

reduction by BacG with delivery of a (pro-S) hydride to C4 and a solvent-derived hydrogen at C3. At the level of these H4HPP keto acid products, and after they had been swept through to the more stable H4-tyrosines via BacF, it is clear that opposite chirality results at C4. The 3Z isomer yields 2S,4S,7R-H4Tyr from BacG action, while the 3E isomer gives 2S,4R,7R-H4Tyr (Figure 6D). We expect the 4S diastereomer is on pathway to anticapsin (4S) but not the 4R diastereomer. However, this proposition must await study of the last two missing enzymatic steps in anticapsin biogenesis. The relative orientation of the dihydronicotinamide ring of the hydride donor NADH and the conjugated dienyl keto acid of the ex-H2HPP must switch between the 3Z and 3E isomers as they are recognized as substrates. If we assume that the NADH orientation stays the same within its BacG binding site, as it transfers the pro-S hydrogen to both geometric isomers of substrate, the different faces of the olefin would be presented to the incoming hydride as shown in panels A and B of Figure 6, assuming the C3−C1 chain of the geometric ex-H2HPP isomers stays in the same orientation.

One expects enzymes to act as chiral catalysts such that of the eight possible H4Tyr diastereomers, only one is anticipated. The 2S,4S diastereomer of H4Tyr is indeed a known nonproteinogenic building block of the cyanobacterial nonribosomal peptides aeruginoside 126A and aeruginoside 126B.22 In the initial structural assignment of anticapsin, the C4 stereochemistry was initially incorrectly assigned1 but corrected subsequent to total synthesis23 as the 2S,4S diastereomer, consistent with 2S,4SH4Tyr being on pathway. The stereochemistry at C7 of anticapsin has become moot because of the oxidation of the C7 alcohol in prephenate to the ketone in anticapsin. Given that we have recently proven that BacA, by isomerizing the pro-R olefin in prephenate, generates the 7R-Δ3,Δ5-diene, we therefore anticipated the H4Tyr emerging from tandem action of BacABGF would thus be the 2S,4S,7R diastereomer. As we have reported here, this expectation is met, but the 2S,4R,7R diastereomer also emerges from the tandem action of the four Bac enzymes. Perhaps most intriguing is the action of the second enzyme, BacB, to generate a mix of 3Z and 3E isomers of ex-H2HPP. Both are then substrates for conjugate hydride 5630

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to obtain the anticapsin warhead: 7R-alcohol oxidation and olefin epoxidation.

The formation of C4 diastereomers of H4HPP (and then of H4Tyr from subsequent BacF action) gives further insight into the possibly corrective role of BacB in the pathway. We have previously noted that the 7R-en-H2HPP product of BacA can undergo nonenzymatic conversion to the more conjugated 7Rex-H2HPP isomer. We wondered why the producing cell would expend energy to make a protein (BacB) that would accelerate a reaction that would otherwise occur. Certainly, matching the kinetics of metabolite flux and prevention of off-pathway decomposition and aromatization are reasonable rationales, but most telling is the finding that the nonenzymatic route gives 98% 3E geometric isomer. The work reported here shows that the E isomer will lead to, via the action of BacG as a reductase, the 4R diastereomer of H4Tyr, which is presumably the wrong chirality to go on to the anticapsin antibiotic. BacB instead equilibrates the more stable 3E and less stable 3Z geometric isomers to give up to 25% of the 3Z isomer, which on subsequent reduction by BacG gives the desired 4S stereochemistry. If there is enough BacB in producer cells coupled to the BacG catalytic efficiency preference of 3Z/E (64/1 found in this study), then flux could be routed selectively to the productive 4S-cyclohexenol scaffold. It may be that the 4R stereochemistry is useful for one or more alternate downstream metabolites in this or related microbes [i.e., the diepi form of 2-carboxy-6-hydroxyoctahydroindole (diepi-Choi) (2R,4R stereochemistry) residues found as constituents of nonribosomal peptides in cyanobacteria].24 While a main objective of this study was to ascertain the C4 stereochemistry from the action of the NADH-utilizing BacG at the stage of reduction of ex-H2HPP to H4HPP, we have also focused on the stereochemical outcomes at C8 and C9 from consecutive action of BacA and BacB in deuterated buffers. While C8 and C9 sequentially become CH2 groups in en-H2HPP and exH2HPP, respectively, and the stereochemistry is thus cryptic, in principle the stereochemical course of each enzyme at those carbons could be revealed with deuterium in place of hydrogen. We have previously found7 that one deuteron from solvent is fixed at C8 and one at C9, and in this study, we have shown that BacA introduces the solvent derived (H+/D+) from above the plane of the diene in prephenate, yielding 6S-en-H2HPP-d1. Because of the numbering priority changes, this compound becomes 8S-ex-H2HPP upon BacB action (Figure 6C). In turn, as gathered from the 4R-H4Tyr result, BacB instead transfers a solvent-derived D+ from below the plane at C9 (H9a) and yields a 9R-deutero product, swept through to H4Tyr. (BacB also removes one of the methylene hydrogens at C3 as it equilibrates the E and Z geometric isomers of ex-H2HPP.8). Interpretation of the 1H−1H NOESY spectrum of protonated 3E-ex-H2HPP (Figure S8B of the Supporting Information) established that H9a is on the ring face opposite of H7. While it will take structural studies of BacA in complex with substrate and/or product to identify candidate side chains of active site residues such as BH+, it must be a very efficient process to derail what has been viewed as the default outcome in other members of the prephenate dehydratase superfamily.16 That default option is cleavage of the C7−O bond as the cyclohexadiene ring aromatizes, and there has been substantial debate over whether decarboxylation and loss of water is a concerted process.25,26 Knowledge of the configuration of the H4Tyr diastereomers generated by the first four enzymes of the Bac pathway, and the realization that 4R and 4S products emerge, will provide the appropriate substrates for evaluation and identification of the enzymes responsible for what should be the two remaining steps

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ASSOCIATED CONTENT

S Supporting Information *

HPLC analysis of ex-H2HPP isomer purity; 1H NMR spectra of 4R- and 4S-2H-NADH; 1H NMR spectra of H4HPP isomers synthesized from 2H-NADH; chiral HPLC analysis of BacFgenerated tyrosine; additional NMR spectra of deuterated forms of ex-H2HPP, H4HPP, and H4Tyr isomers; and tabulated NMR spectral data for H4HPP and H4Tyr isomers. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (617) 432-1715. Fax: (617) 432-0483. Funding

This work was supported in part by National Institutes of Health Grants AI042738 and GM49338 (C.T.W.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Drs. Sarah A. Mahlstedt, Stuart W. Haynes, Timothy A. Wencewicz, and Steven J. Malcolmson for helpful advice and discussions.

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ABBREVIATIONS H2HPP, dihydro-4-hydroxyphenylpyruvate; H4HPP, tetrahydro4-hydroxyphenylpyruvate; H4Tyr, tetrahydrotyrosine; HPP, 4hydroxyphenylpyruvate; LC−MS, liquid chromatography−mass spectrometry; NMR, nuclear magnetic resonance; gCOSY, gradient homonuclear correlation spectroscopy; gHSQC, gradient heteronuclear single-quantum coherence; gHMBC, gradient heteronuclear multiple-bond coherence; NOESY, nuclear Overhauser effect spectroscopy; HPLC, high-performance liquid chromatography; D2O, deuterium oxide; GlcNAc, Nacetylglucosamine.

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REFERENCES

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